Combining iRGD with HuFOLactis enhances antitumor potency by facilitating immune cell infiltration and activation

Abstract

Multiple research studies have demonstrated the efficacy of lactic acid bacteria in boosting both innate and adaptive immune responses. We have created a Lactococcus lactis variant that produces a modified combination protein with Fms-like tyrosine kinase 3 ligand and co-stimulator O × 40 ligand, known as HuFOLactis. The genetically modified variant was purposely created to activate T cells, NK cells, and DC cells in a laboratory setting. Furthermore, we explored the possibility of using the tumor-penetrating peptide iRGD to deliver HuFOLactis-activated immune cells to hard-to-reach tumor areas. Following brief stimulation with HuFOLactis, immune cell phenotypes and functions were assessed using flow cytometry. Confocal microscopy was employed to demonstrate the infiltrative and cytotoxic capabilities of iRGD-modified HuFOLactis-activated immune cells within tumor spheroids. The efficacy of iRGD modified HuFOLactis-activated immune cells against tumors was assessed in xenograft mouse models. HuFOLactis treatment resulted in notable immune cell activation, demonstrated by elevated levels of CD25, CD69, and CD137. Additionally, these activated immune cells showed heightened cytokine production and enhanced cytotoxicity against MKN45 cell lines. Incorporation of the iRGD modification facilitated the infiltration of HuFOLactis-activated immune cells into multicellular spheroids (MCSs). Additionally, immune cells activated by HuFOLactis and modified with iRGD, in combination with anti-PD-1 treatment, effectively halted tumor growth and prolonged survival in a mouse model of gastric cancer.

Keywords: Fms-like tyrosine kinase 3 ligand; Lactococcus lactis; OX40 ligand; iRGD; immunotherapy.

Figure 1.

HuFOLactis was characterized and it was found to be effective in boosting the activation of DCs. (a) The schematic shows intravenous injection of PBMCs modified with iRGD and activated with HuFOLactis, along with a PD-1 mAb, for improved tumor immunotherapy. Created with MedPeer. (b) The quantification of the target protein in the bacterial lysates of HuFOLactis was carried out. Bacteria (10⁹ CFU) were collected and then sonicated to isolate the pellets. The assessment of the target protein levels in the bacterial lysates of HuFOLactis was conducted through ELISA (n = 5 biologically independent samples). (c) Analysis of the colocalization of HuFOLactis (DiO; green) in PBMC-derived DCs (DiI; red) was conducted using confocal microscopy after a two-hour incubation period. We depicted a representative out of three independent experiments yielding similar results. (d) Representative flow plots illustrating the expression levels of CD80, CD86, and HLA-DR on CD11c⁺ DC cells following a 48-hour in vitro co-incubation with Lactis or HuFOLactis are presented. (e) Summary of data from D showing CD80⁺, CD86⁺, and HLA-DR⁺ cells among CD11c⁺ cells (mean ± s.e.m.; n = 3 cell cultures per group). (f) IL-12p70 concentrations in DCs supernatants (mean ± s.e.m.; n = 3 cell cultures per group). (g) The influence of inactivated HuFOLactis on IL-12p70 secretion and CD80 upregulation in dendritic cells (mean ± s.e.m.; n = 3 cell cultures per group). Data were analyzed by one-way ANOVA coupled with Tukey’s multiple-comparisons test. For experiments C-G, three independent experiments were performed using PBMCs from three donors, with three cell cultures per group in each experiment. The displayed result is representative of one of these three independent experiments.

Figure 2.

After co-culturing PBMCs with HuFOLactis, both T cells and NK cells can be activated. (a, b) Representative flow plots showing the expression of CD25, CD69, and CD137 on CD3⁺ T cells and CD56⁺ NK cells after co-incubation with Lactis or HuFOLactis in vitro for 48 hours. (c) Summary of data from a showing CD25⁺, CD69⁺, and CD137⁺ cells among CD3⁺ T cells (mean ± s.e.m.; n = 3 cell cultures per group). (d) Summary of data from B showing CD25⁺, CD69⁺, and CD137⁺ cells among CD56⁺ NK cells (mean ± s.e.m.; n = 3 cell cultures per group). (e) Assessment of Th1/Th2 in coculture supernatants after PBMCs stimulated by Lactis or HuFOLactis for 48 hours in vitro (mean ± s.e.m.; n = 3 cell cultures per group). Data were analyzed by one-way ANOVA coupled with Tukey’s multiple-comparisons test. Three independent experiments were performed using PBMCs from three donors, with three cell cultures per group in each experiment. The displayed result is representative of one of these three independent experiments.

Figure 3.

HuFOLactis enhance the induction of oncoprotein-specific T cells. PBMCs were co-cultured with crude lysates from HuFOLactis or lactis, as well as MKN45 tumor cell membranes (TM). After 14 days of culture, cells were harvested and tested for their ability to produce IFN-γ and TNF-α in response to the MKN45 cells. (a, e) Representative flow plots and graphs showing the expression of IFN-γ on CD4⁺ and CD8⁺ T cells (mean ± s.e.m.; n = 3 cell cultures per group). (b, f) Representative flow plots and graphs showing the expression of TNF-α on CD4⁺ and CD8⁺ T cells (mean ± s.e.m.; n = 3 cell cultures per group). (c, g) Representative flow plots and graphs showing the Tregs in the final cultured cell product (mean ± s.e.m.; n = 3 cell cultures per group). (d, h) Representative flow plots and graphs showing central memory T cells in the final cultured cell product (mean ± s.e.m.; n = 3 cell cultures per group). Data were analyzed by one-way ANOVA coupled with Tukey’s multiple-comparisons test. Three independent experiments were performed using PBMCs from three donors, with three cell cultures per group in each experiment. The displayed result is representative of one of these three independent experiments.

Figure 4.

HuFOLactis activated PBMCs modified with iRGD possess superior penetration capacity in MCSs. (a) Representative morphological assessment of HGC27-MCSs was exposed to indicated CFSE stained HuFOLactis activated PBMCs at an effector to target cell ratio (E:T) of 5:1 calculated on the initial number of spheroids inoculated for 6 h before confocal microscopy. (b) Summary of data demonstrates the depth of infiltration of HGC27-MCSs by specific cells over a 6-hour period through quantitative analysis of mean fluorescence intensity (mean ± s.e.m.; n = 4 MCSs per group). (c, d) the cytotoxic reactivity of HuFOLactis activated PBMCs was measured using CFSE/PI cytotoxicity assay, the target cells were MKN45 and HGC27, respectively (mean ± s.e.m.; n = 3 cell cultures per group). (e, f) Flow cytometry was used to assess PD-L1 expression after 48 hours of culturing the MKN45 and HGC27 cell lines with HuFOLactis activated PBMCs, at an E: T ratio of 20:1 (mean ± s.e.m.; n = 3 cell cultures per group). For experiments B, data were analyzed by one-way ANOVA coupled with Tukey’s multiple-comparisons test. For experiments C and D, Data were analyzed by two-way ANOVA coupled with Tukey’s multiple-comparisons test. For experiments E and F, Data were analyzed by two-sided unpaired t-test. Three independent experiments were conducted with PBMCs from three donors, with four MCSs or three cell cultures per group in each experiment. A representative result is shown from one of the three independent experiments.

Figure 5.

The combination of iRGD with HuFOLactis and anti-PD1 antibody improved mouse survival. (a) Initiating an antitumor experiment in vivo, five-week-old female Babl/c-nude mice were injected with 5 × 10⁶ MKN45 cells subcutaneously. Two weeks later, 2 × 10⁷ HuFO-PBMCs (activated with HuFOLactis for 24 h) either modified or not with iRGD were injected into the mice. At the same time, two groups were given a 250 µg intravenous PD-1 blockade. The observation of tumor burden and survival time in the mice was conducted. (b) Average tumor-growth curves of mice bearing MKN45 tumor with different treatments as indicated (mean ± s.e.m.; n = 10 biologically independent mice per group) (mean ± s.e.m.; n = 10 biologically independent mice per group). (c) Survival curves of mice in different groups for 80 days (n = 9 biologically independent mice per group). (d, e) Flow cytometry was employed to analyze the percentage of human CD3⁺ T cells and CD11c⁺ DCs in the tumor tissue of HuFO-PBMC with or without iRGD treatment in mice, seven days after intravenous injection of cells. (f, g) Summary of data from D and E (mean ± s.e.m.; n = 5 biologically independent mice per group). (h) The average weight of different groups for 30 days (mean ± s.e.m.; n = 6 biologically independent mice per group). (i, j) Flow cytometric analysis polyfunctional CD3⁺ T cells with positive staining for IFN-γ and exhausted CD3⁺ T cells with Tim-3 expression. (K, L) Quantifications of IFN-γ and TIM-3 expression in CD3⁺ T cells (mean ± s.e.m.; n = 4 biologically independent mice per group). For experiments B, p-values were determined by one-way ANOVA with Tukey’s multiple comparisons test. Differences in survival were determined by using the Kaplan–Meier method, and the p value was determined via the log-rank (Mantel–Cox) test. For experiments F, G, K and L, data were analyzed using one-way ANOVA and each data point represents one sample from an independent mouse. Animal experiments were repeated twice under similar conditions with similar results.